# Exploiting simultaneous positive and negative selection to advance directed evolution of orthogonal RNA-guided nucleases

> **NIH NIH F32** · NEW YORK UNIVERSITY SCHOOL OF MEDICINE · 2021 · $68,562

## Abstract

Project Summary
Programmable RNA-guided nucleases are facilitating top-down genome editing endeavors throughout the
biomedical research community, and show great potential for use in therapeutic applications. The sequence
specificity of commonly used RNA-guided nucleases, including Streptococcus pyogenes Cas9 (SpCas9), is
jointly enabled by RNA-DNA base pairing and essential interactions with a target-abutting DNA sequence
known as a protospacer adjacent motif (PAM). Although SpCas9 can accommodate virtually any guide RNA
sequence, its sequence-specific interactions with PAMs are invariably determined by protein-DNA contacts.
SpCas9’s overall programmability is therefore constrained by its intrinsic PAM requirement, and this can limit
editing applications that require very precise target site positioning.
Previous studies have employed procedures for protein engineering, including directed evolution, to alter the
protein component of RNA-guided nucleases such as SpCas9 in various ways. While not exhaustive, some of
these studies have successfully produced variants of SpCas9 with certain alternative PAM specificities. Among
these, the directed evolution regimens tested to date involved custom microbial systems engineered to impose
positive selection on SpCas9’s PAM specificity in vivo. Positive selection is sufficient to drive the evolution of
protein variants with relaxed specificity, as well as variants with truly altered, orthogonal specificity. Whereas
relaxed PAM specificities that broaden the genomic sequence space accessible for targeting may be ideal for
typical editing applications, orthogonal variants are still of use in certain allele-specific editing applications
where the PAM requirement can be exploited to discriminate between two alleles that differ by only a single
nucleotide. To favor the evolution of orthogonal variants, directed evolution procedures may incorporate
negative selection pressures that counterselect against variants which retain their parental substrate
preferences. Whether such strategies would be effective for engineering orthogonal PAM specificities has not
been investigated, however.
The goal of this proposed study is to establish and evaluate microbial directed evolution regimens that impose
simultaneous positive and negative selection, or positive selection alone, on SpCas9’s PAM specificity. Side-
by-side evaluation of these procedures will be enabled by high-throughput profiling of variant pools evolved in
the absence or presence of negative selection, along with mutational dissections and clonal benchmarking
assays. Collectively, these experiments will ensure identification of the most desirable SpCas9 variants, and
guide the systematic refinement of directed evolution procedures intended to generate orthogonal protein
functions.

## Key facts

- **NIH application ID:** 10133453
- **Project number:** 5F32GM137482-02
- **Recipient organization:** NEW YORK UNIVERSITY SCHOOL OF MEDICINE
- **Principal Investigator:** Gregory William Goldberg
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $68,562
- **Award type:** 5
- **Project period:** 2020-04-01 → 2022-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10133453

## Citation

> US National Institutes of Health, RePORTER application 10133453, Exploiting simultaneous positive and negative selection to advance directed evolution of orthogonal RNA-guided nucleases (5F32GM137482-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10133453. Licensed CC0.

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